Derivatives of sulindac can protect normal cells against oxidative damage

ABSTRACT

The disclosure provides chemical compounds possessing therapeutic and/or protective properties against oxidative damage. Methods of making such therapeutic and/or protective compounds and associated compositions are also provided, as are methods for their use, which include protecting cells from oxidative damage and/or inhibiting production of ROS in a cell or subject, as well as preventing or reducing the extent of tissue damage caused by an ischemic event in a subject at elevated risk of such an event.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase entry under 35 U.S.C. § 371 ofInternational Application No. PCT/US2018/022885, filed Mar. 16, 2017,published in English, which claims the benefit of priority under 35U.S.C. § 119(e) to U.S. Provisional Application No. 62/472,785, filed onMar. 17, 2017, entitled, “Derivatives of Sulindac Can Protect NormalCells Against Oxidative Damage”. The contents of these relatedapplications are incorporated herein by reference in their entireties.

GOVERNMENTAL SUPPORT

Development of the present invention was supported, at least in part, bythe National Cancer Institute of the National Institutes of Health underAward Number 1R01CA131378. Therefore, the Government may have certainrights in the invention.

BACKGROUND OF THE INVENTION

Oxygen is involved in a wide range of normal metabolic reactions and isessential for the survival of all aerobic organisms, including humanbeings. Reactive oxygen species (ROS), such as superoxide, are producedin abundance as a byproduct of the incomplete reduction of oxygen thathas entered the respiratory chain. Superoxide is the precursor of otherdamaging oxygen species includes hydrogen peroxide, the hypochlorite ionand the hydroxyl radical. Oxidase enzymes in cells such as phagocytesand nitric oxide synthases are other sources of ROS.

While low levels of ROS are present under normal physiologicalconditions, in excess, ROS can cause oxidative damage to cells andtissues by, for example, oxidizing cellular macromolecules such asnucleic acids, lipids and proteins. Cumulative damage to cells in thismanner can result in pathology. Not surprisingly, oxidative damage hasbeen implicated in a wide variety of diseases and conditions includingchronic obstructive lung disorders such as smoker's emphysema,reperfusion disease, neurodegenerative diseases such as Alzheimer'sdisease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS),heart attacks, stroke, several autoimmune diseases and cancer.

The balance between the cellular production of reactive oxygen species(ROS), primarily from mitochondrial respiration, and the ability ofcells to protect against oxidative damage very likely also determinesthe rate and extent of the normal aging process in humans and othermammals. Our laboratory initially became interested in the role ofcellular protective mechanisms against oxidative damage afteridentifying methionine sulfoxide reductase A (MsrA), the first member ofthe Msr family of enzymes (1). MsrA reduces methionine sulfoxideresidues (S epimer) in proteins back to methionine, repairing oxidativedamage. The other major Msr family member is MsrB, which reduces the Repimer of methionine sulfoxide in proteins (2). The Msr system is alsopart of a ROS scavenger system that permits methionine residues inproteins to function as catalytic anti-oxidants (3). Overexpression ofmammalian MsrA in D. melanogaster was reported to extend their lifespan(4), while MsrA has been shown to be involved in the extension of thelifespan of C. elegans mutants lacking the daf 2 gene (5) and on calorierestricted diets (6).

Small molecules that contain a methyl sulfoxide moiety, such as theanti-inflammatory drug sulindac, can also serve as substrates for theMsr enzymes. It is known that MsrA can reduce the S epimer of sulindac.Recently, we have described a protein purified from liver extracts thatcan reduce the R epimer of sulindac (7). As shown in FIG. 1, sulindac isa mixture of both epimers and a pro drug containing a methyl sulfoxidegroup that can be converted by these enzymes to sulindac sulfide, theactive metabolite of sulindac responsible for its cyclooxygenase COXinhibitory activity (7). Initial studies with sulindac also showedunexpected properties of this drug. For example, sulindac atconcentrations that were not cytotoxic, could sensitize cancer cells tooxidizing agents, resulting in enhanced anticancer activity (8).However, under similar conditions sulindac protected normal lung cellsagainst oxidative damage by a mechanism that was independent of its COXinhibitory activity or the Msr system (8). As shown in further studiesof ischemia/reperfusion damage to the heart (9) and oxidative and UVdamage to retinal pigmented epithelial (RPE) cells (10), sulindac isable to initiate a protective pharmacological preconditioning responseby a mechanism similar to the well documented protective mechanism,referred to as ischemic preconditioning (IPC)(11). However, unlike IPCwhich is initiated by sub-lethal hypoxic conditions, the protectiveeffect of sulindac was obtained under normoxic conditions, which werefer to as pharmacological preconditioning.

Long term uses of nonsteroidal anti-inflammatory drugs (NSAIDS) such assulindac are limited by gastrointestinal, renal and cardiovasculartoxicities, which are caused by the inhibition of COX-1 and COX-2 andsuppression of physiologically important prostaglandins. Sulindacsulfone, a metabolic oxidation product of sulindac, has no COXinhibitory activity, but is more active than sulindac in protectingcells against oxidative damage, which suggests that the cytoprotectivemechanism is not related to the COX inhibitory activity of sulindac (8,10). However, sulindac sulfone is significantly more toxic to cellsrelative to sulindac.

Thus, it would be advantageous to make sulindac derivatives that arenon-cytotoxic, but more potent in protecting cells against oxidativedamage and that lack COX inhibitory activity.

SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to chemicalcompounds, represented by formulas (I) and (II), herein, orpharmaceutically acceptable salts thereof. A second aspect of thepresent invention is directed to methods of making the chemicalcompounds. A third aspect of the present invention is directed topharmaceutical compositions containing an effective amount of a compoundof formula (I) or (II), and optionally a pharmaceutically acceptablecarrier. A fourth aspect of the present invention is directed to amethod of treating a subject with a disease or condition the cause orprogression of which involves the production of reactive oxygen species(ROS) and resultant oxidative stress, which entails administering to thesubject a therapeutically effective amount of a compound of formula (I)or (II). A related aspect is directed to a method for protecting cellsin a subject from oxidative damage, e.g., caused by a reperfusioninjury, which entails administering to the subject an effective amountof a compound of formula (I) or (II). A further aspect is directed to amethod of inhibiting production of ROS in a cell, in vivo or in vitro,that involves contacting the cell with an effective amount of a compoundof formula (I) or (II). A further aspect is directed to a method ofprotecting normal cells against oxidative damage, in vivo or in vitro,e.g., by environmental factors such as UV irradiation or diseases, thatinvolves contacting the cell with effective amount of a compound offormula (I) or (II).

An additional aspect of the present invention provides a method ofpreventing or reducing the extent of tissue damage of an ischemic eventin a subject at elevated risk of an ischemic event, the method involvingidentifying a subject at elevated risk of an ischemic event andadministering a therapeutically effective amount of the compound(Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)aceticacid, or a pharmaceutically acceptable salt or prodrug thereof to thesubject, thereby preventing or reducing the extent of tissue damage ofan ischemic event in the subject identified as being at elevated risk ofan ischemic event.

In one embodiment, the subject possesses one or more of the followingrisk factors for an ischemic event: high blood pressure, heart disease,high cholesterol levels, sleep apnea, previous occurrence of stroke,smoking, excessive alcohol consumption and excessive weight. Optionally,the ischemic event is an ischemic event of a tissue selected from thegroup consisting of the heart, brain, kidneys and liver.

In certain embodiments, the method further involves administering to thesubject a therapeutically effective amount of aspirin, a blood pressuremedication, a type II diabetes medication and/or an mTOR inhibitor. Inone embodiment, the type II diabetes medication is metformin. In certainembodiments,(Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)aceticacid, or a pharmaceutically acceptable salt or prodrug thereof and theaspirin, blood pressure medication, type II diabetes medication and/ormTOR inhibitor are co-administered to the subject

Compounds of the present invention, e.g., Compound 9 as describedherein, are significantly more potent than sulindac in protecting cellsfrom oxidative damage, and exhibit essentially no COX inhibitoryactivity. Without intending to be bound to any particular theory ofoperation, the mechanism of action of the inventive compounds may berelated to their ability to inhibit cyclic nucleotide degradingphosphodiesterase (PDE) isozymes such as PDE5 and/or PDE10, resulting inan increase in intracellular cGMP and/or cAMP levels that could initiatea pharmacological preconditioning response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of sulindac and its metabolites. MsrA andMsrB1 can reduce the S and R epimers of sulindac, respectively, tosulindac sulfide, the active COX inhibitor. The oxidation of the epimersis catalyzed by members of the cytochrome (CYP)-P-450 system.

FIG. 2 shows protection of RPE cells against oxidative damage from TBHPby sulindac, Compound 9 and Compound 10. Sulindac was tested at aconcentration of 200 while inventive Compounds 9 and 10 were tested at aconcentration of 25 μM.

FIG. 3 shows concentration-dependent protection of RPE cells againstoxidative damage from TBHP by Compound 9.

FIG. 4 shows that Compound 9 lacks significant COX inhibitory activity.FIG. 4A shows that Compound 9 is at least 20× less potent than the knownCOX-1 inhibitor, sulindac sulfide (SS). FIG. 4B shows that Compound 9 isapproximately 1000× less potent than the known COX-2 inhibitor,celecoxib.

FIG. 5 shows PDE inhibitory activity of Compound 9. FIG. 5A showsconcentration-dependent inhibition of PDE5 by Compound 9. FIG. 5B showsconcentration-dependent inhibition of PDE10 by Compound 9. FIG. 5C showsthe ability of Compound 9 to inhibit other PDE isozymes when testedagainst a panel of PDE isozymes at a 25 μM concentration.

FIG. 6 shows the protective effect of Compound 9 on RPE cells through apreconditioning mechanism. RPE cells were incubated with: (A) ROSscavenger, tiron (disodium 4,5 dihydroxy-1,3 benzenedisulfonate) (1 mM);(B) PKC inhibitor (PKCI) chelerythrine (2 μM); or the (C) PKG inhibitor,Rp-Br-8-PET-cGMPS (500 μM) for 24 h in combination with Compound 9.

FIG. 7 demonstrates the protective effect of administering Compound 9before performing the Langendorff procedure. Animals were administeredeither no drug or Compound 9 (0.7 mg/kg daily for 48 h) before isolationof the heart for analysis on the Langendorff apparatus. Total LDH levels(increased LDH levels indicating decreased cardiac cell viability) areshown after 45 min ischemia and 2 h of reperfusion, respectively (n=1animal administered Compound 9, as compared to four control (nocompound) animals).

FIGS. 8A and 8B demonstrate infarct sizes of hearts as measured bytriphenyltetrazolium chloride (TTC) staining. FIG. 8A shows resultsobtained for hearts obtained from rats initially administered no drug orCompound 9, exposed to ischemia and reperfusion, stained with 1% TTC,then cut transversely into 2-mm sections. Percent infarction wasdetermined using NIH image J analysis software. Histograms representpercent of heart that was infarcted. (n=one animal administered Compound9, as compared to four animals administered no compund). FIG. 8B showsphotographs of representative sections from Langendorff hearts subjectedto TTC staining following 45 min ischemia and 2 h reperfusion, showinginfarcted tissue (white) and viable tissue (red).

FIG. 9 shows that Compound 9 administered via i.p. injection at 0.7mg/kg demonstrated a robust protective effect both during the 45 minischemia phase of the Langendorff procedure and during the 2 hreperfusion phase. Sulindac exhibited no protective effect during the 45min ischemia phase when administered at 0.7 mg/kg i.p. daily for 48 hprior to the Langendorff procedure, and the protective effect that wasobserved for sulindac during the 2 h reperfusion phase was not as robustas that observed for Compound 9.

DETAILED DESCRIPTION

I. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below.

All publications, patents, and other references mentioned herein areincorporated by reference in their entirety.

The nomenclature used in this Application is based on IUPAC systematicnomenclature, unless indicated otherwise.

Any open valency appearing on a carbon, oxygen, sulfur or nitrogen atomsin the structures herein indicates the presence of a hydrogen, unlessindicated otherwise.

When indicating the numbers of substituents, the term “one or more”refers to the range from one substituent to the highest possible numberof substitution, i.e., replacement of one hydrogen up to replacement ofall hydrogens by substituents.

The term “substituent” denotes an atom or a group of atoms replacing ahydrogen atom on the parent molecule.

The term “substituted” denotes that a specified group bears one or moresubstituents. Where any group can carry multiple substituents and avariety of possible substituents is provided, the substituents areindependently selected and need not to be the same. The term“unsubstituted” means that the specified group bears no substituents.The term “optionally substituted” means that the specified group isunsubstituted or substituted by one or more substituents, independentlychosen from the group of possible substituents. When indicating thenumber of substituents, the term “one or more” means from onesubstituent to the highest possible number of substitution, i.e.,replacement of one hydrogen up to replacement of all hydrogens bysubstituents.

The term “pharmaceutically acceptable acid addition salt” denotes thosepharmaceutically acceptable salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,carbonic acid, phosphoric acid, and organic acids selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic, and sulfonic classes of organic acids such as formic acid,acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid,pyruvic acid, oxalic acid, malic acid, maleic acid, maloneic acid,succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid,ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamicacid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonicacid, ethanesulfonic acid, p-toluenesulfonic acid, and salicyclic acid.

The term “halo”, “halogen”, and “halide” are used interchangeably hereinand denote fluorine, chlorine, bromine and iodine.

The term “alkyl” denotes a monovalent linear or branched saturatedhydrocarbon group of 1 to 12 carbon atoms. In particular embodiments,alkyl has 1 to 7 carbon atoms, and in some embodiments 1 to 4 carbonatoms, and in yet other embodiments, 1, 2 or 3 carbon atoms. Examples ofalkyl include methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl,sec-butyl, or tert-butyl, and particularly methyl.

The term “alkenyl denotes a monovalent linear or branched hydrocarbongroup of 2 to 7 carbon atoms with at least one double bond. Inparticular embodiments, alkenyl has 2 to 4 carbon atoms with at leastone double bond. Examples of alkenyl include ethenyl, propenyl,prop-2-enyl, isopropenyl, n-butenyl, and iso-butenyl.

The term “alkynyl” denotes a monovalent linear or branched saturatedhydrocarbon group of 2 to 7 carbon atoms comprising one, two or threetriple bonds. In particular embodiment's alkynyl has from 2 to 4 carbonatoms comprising one or two triple bonds. Examples of alkynyl includeethynyl, propynl, prop-2-ynyl, isopropynyl, and n-butynyl.

The term “alkoxy” denotes a group of the formula —O—R′, wherein R′ is analkyl group. Examples of alkoxy moieties include methoxy, ethoxy,isopropoxy, and tert-butoxy, and particularly methoxy.

The term “carbocyclic” denotes a monovalent saturated or aromaticmonocyclic or bicyclic hydrocarbon group of 3 to 10 ring carbon atoms.In particular embodiments cycloalkyl denotes a monovalent saturatedmonocyclic hydrocarbon group of 3 to 8 ring carbon atoms. Bicyclic meansconsisting of two saturated carbocycles having one or more carbon atomsin common. Particular cycloalkyl groups are monocyclic. Examples formonocyclic cycloalkyl are cyclopropyl, cyclobutanyl, cyclopentyl,cyclohexyl or cycloheptyl. Examples for bicyclic cycloakyl arebicyclo[2.2.1]heptanyl, or bicyclo[2.2.2]octanyl.

The term “heterocyclic” denotes a monovalent saturated or partlyunsaturated aromatic monocyclic ring system of 3 to 9 ring atoms,comprising 1, 2, or 3 ring heteroatoms selected from N, O and S, theremaining ring atoms being carbon. In particular embodiments,heterocycloalkyl is a monovalent saturated monocyclic ring system of 4to 7 ring atoms, comprising 1, 2, or 3 ring heteroatoms selected from N,O and S, the remaining ring atoms being carbon. Examples for monocyclicsaturated heterocycloalkyl are aziridinyl, oxiranyl, azetidinyl,oxetanyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydro-thienyl,pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl,thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl,piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl,azepanyl, diazepanyl, homopiperazinyl, and oxazepanyl.

The term “aromatic” denotes the conventional idea of aromaticity asdefined in the literature, in particular in IUPAC—Compendium of ChemicalTerminology, 2nd, A. D. McNaught & A. Wilkinson (Eds). BlackwellScientific Publications, Oxford (1997).

The term “aryl” denotes a monovalent aromatic carbocyclic mono- orbicyclic ring system comprising 6 to 10 carbon ring atoms. Examples ofaryl moieties include phenyl and naphthyl.

The term “aryl(alkyl)oxy” denotes a group of the formula —O—(CH₂)_(n)—R′wherein R′ is aryl (e.g., phenyl), and n is 0, 1 or 2. An example ofarylalkyloxy is benzyloxy.

The term “heteroaryl” denotes a monovalent aromatic heterocyclic mono-or bicyclic ring system of 5 to 12 ring atoms, comprising 1, 2, 3 or 4heteroatoms selected from N, O and S, the remaining ring atoms beingcarbon. Examples of heteroaryl moieties include pyrrolyl, furanyl,thienyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl,thiadiazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyrazolyl, pyridazinyl,pyrimidinyl, triazinyl, azepinyl, diazepinyl, isoxazolyl, benzofuranyl,isothiazolyl, benzothienyl, indolyl, isoindolyl, isobenzofuranyl,benzimidazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl,benzoisothiazolyl, benzooxadiazolyl, benzothiadiazolyl, benzotriazolyl,purinyl, quinolinyl, isoquinolinyl, quinazolinyl, or quinoxalinyl, mostparticularly pyrazolyl and pyridinyl.

The term “alkylene” denotes a linear saturated divalent hydrocarbongroup of 1 to 7, e.g., 1-2 or 1-3 carbons, carbon atoms or a divalentbranched saturated divalent hydrocarbon group of 3 to 7 carbon atoms.Examples of alkylene groups include methylene, ethylene, propylene,2-methylpropylene, butylene, 2-ethylbutylene, pentylene and hexylene.

The term “alkylamino” denotes a group —NR′R″, wherein R′ is hydrogen andR″ is an alkyl, e.g., C1-3 alkyl such methyl. The term “dialkylamino” asused herein denotes a group —NR′R″, wherein R′ and R″ are both alkyl.Examples of alkylamino groups include methylamino and ethylamino.Examples of alkylamino groups include dimethylamino, methylethylamino,diethyl amino and di(1-methylethyl)amino.

The terms “pharmaceutical composition” and “pharmaceutical formulation”(or “formulation”) are used interchangeably and denote a mixture orsolution comprising a therapeutically effective amount of an activepharmaceutical ingredient together with one or more pharmaceuticallyacceptable carriers to be administered to a subject, e.g., a human inneed thereof.

The term “pharmaceutically acceptable” denotes an attribute of amaterial which is useful in preparing a pharmaceutical composition thatis generally safe, non-toxic, and neither biologically nor otherwiseundesirable and is acceptable for veterinary as well as humanpharmaceutical use.

When the compounds of this invention are solids, it is understood bythose skilled in the art that the compounds described herein, and theirsolvates and salts, may exist in different solid forms, particularlydifferent crystal forms, all of which are intended to be within thescope of the present invention and specified formulas.

The term “pharmaceutically acceptable salts” denotes salts which are notbiologically or otherwise undesirable. Pharmaceutically acceptable saltsinclude acid addition salts. The terms “pharmaceutically acceptableexcipient” and “pharmaceutically acceptable carrier” denote anypharmaceutically acceptable ingredient in a pharmaceutical compositionthat is physiologically acceptable and, has no therapeutic activity, andis non-toxic to the subject administered and inert with respect to thecompound of formulas (I) and (II), and any other active agent that maybe present in the composition.

A “subject” is a mammal. Mammals include domesticated animals (e.g.,cows, sheep, cats, dogs, and equines such as horses), primates (e.g.,humans and non-human primates such as monkeys), rabbits, and rodents(e.g., mice and rats). In certain embodiments, the subject is a human.

As used herein “amelioration” or “treatment” (or “treating”) isunderstood as meaning to lessen or decrease at least one sign, symptom,indication, or effect of a specific disease or condition. For example,amelioration or treatment of retinitis pigmentosa (RP) can be to reduce,delay, or eliminate one or more signs or symptoms of RP such as areduction in night vision, a reduction in overall visual acuity, areduction in visual field, a reduction in the cone density in one ormore quadrants of the retina, thinning of retina, particularly the outernuclear layer, reduction in a- or b-wave amplitudes on scotopic orphotopic electroretinograms (ERGs); or any other clinically acceptableindicators of a disease state or progression thereof. Amelioration andtreatment can require the administration of more than one dose of acompound of formula (I) or (II), either alone or in conjunction withother therapeutic agents and interventions. Amelioration or treatmentdoes not require that the disease or condition be cured.

As used herein, “prevention” is understood as to limit, reduce the rateor degree of onset, or inhibit the development of at least one sign orsymptom of a disease or condition particularly in a subject prone todeveloping the disease or condition. For example, a subject having amutation in a gene, such as the opsin gene, is likely to developretinitis pigmentosa (RP). The age of onset of one or more symptoms ofthe disease can sometimes be determined by the specific mutation.Prevention can thus include the delay of onset of one or more signs orsymptoms of RP and need not be prevention of appearance of at least onesign or symptom of the disease throughout the lifetime of the subject.

As used herein, “protecting” or “protection” of cells including normalcells, refers to any oxidative damage a cell can sustain, in particular,oxidative damage from environmental factors, for example, the sun,carcinogens; diseases, such as neurological diseases, cancer, drugs suchas adriamycin and arsenic trioxide, diseases caused by organisms, suchas viruses, bacteria and the like. Protecting against oxidative damagecomprises preventing, inhibiting, reducing, reactive oxidationintermediates which cause damage to a cell.

The terms “therapeutically effective amount, “effective amount,” and“effective dose” refer to that amount of a compound of formula (I) and(II) to produce the intended pharmacological, protective, therapeutic orpreventive result. The pharmacologically effective amount may result inthe prolonged survival, amelioration of one or more signs or symptoms ofa disease or condition, or inhibition or reduction of cellularproduction of ROS in vivo or in vitro. For example, in the context ofthe treatment of an ocular disease or condition, a therapeuticallyeffective amount preferably refers to the amount of a compound offormula (I or II) that decreases the loss of night vision, the loss ofoverall visual acuity, the loss of visual field, by at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or more as compared to an untreated controlsubject over a defined period of time, e.g., 2 weeks, one month, 2months, 3 months, 6 months, one year, 2 years, 5 years, or longer. Morethan one dose may be required to provide an effective dose.

II. Inventive Compounds

A first aspect of the present invention is directed to novelcompositions of matter. In some embodiments, the compounds arerepresented by formula (I), as follows:

-   wherein: R, R₀, R₁, R₂, R₃, R₄, R₁₁, and R₁₂ are independently    selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy,    alkylmercapto, alkylsulfoxide, alkyl sulphone, hydroxy, amino,    alkyamino, dialkylamino, azido, cyano, halogen, and substituted or    unsubstituted aryl or substituted or unsubstituted aryl(alkyl)oxy;-   wherein R₅, R₆, R₇, R₈, R₉ and R₁₀, if present, are independently    selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy,    alkylmercapto, alkylsulfoxide, alkyl sulphone, hydroxy, amino,    alkyamino, dialkylamino, azido, cyano, halogen, oxide, and    substituted or unsubstituted aryl or substituted or unsubstituted    aryl(alkyl)oxy; or wherein any neighboring two of R to R₁₂ may form    a saturated or unsaturated, optionally substituted carbocyclic or    heterocyclic ring;-   X represents hydrogen, hydroxyl, substituted or unsubstituted amino,    substituted or unsubstituted alkoxyamino, substituted or    unsubstituted alkyloxy, substituted or unsubstituted thiol,    substituted or unsubstituted alkylmercapto, substituted or    unsubstituted alkyl; or when Y and Y′ together represent O, X    represents a substituted amine or a group A-(CH₂)_(m)—R₁₃, wherein A    represents O or N, R₁₃ is a saturated or unsaturated, substituted or    unsubstituted carbocyclic or heterocyclic group containing at least    one hetero atom which is nitrogen, oxygen, or sulfur, and wherein    the substituents are independently selected from alkyl, alkoxy,    alkylmercapto, alkylsulfoxide, alkylsulphone, cyano, halogen,    hydroxy, amino, alkyamino, dialkylamino, azido, and substituted or    unsubstituted benzyl, and when R₁₃ represents substituted or    unsubstituted aryl, the aryl substituents may be alkyl, alkyloxyl,    alkylmercapto, amino, hydroxyl or halogen; and wherein m is 0, 1 or    2;-   Y and Y′ are independently selected from hydrogen, hydroxyl, or    wherein Y and Y′ together may be a carbon, oxygen, sulfur, or a    nitrogen atom;-   Z represents carbon, nitrogen, oxygen or sulfur;-   and n is 0, 1 or 2; or a pharmaceutically acceptable salt thereof.

In some embodiments, R and R₀ are each hydrogen. In some embodiments, atleast one of R₁-R₄ is alkyloxy, e.g., methoxy, and in some otherembodiments, R₂ is alkoxy or both R₂ and R₃ are alkyloxy. In someembodiments, R₇ is hydrogen or halogen, e.g., F or Cl. In someembodiments, at least one of R₇, R₈ and R₉ is alkyloxy, e.g., methoxy,and R₈ is a substituted aryl(alkyl)oxy group, wherein the substituent isalkyloxy, e.g., methoxy, and in some other embodiments, R₇ and R₉ areeach alkoxy, and in yet other embodiments, each of R₇, R₈ and R₉ isalkyloxy. In some embodiments, R₁₁ is hydrogen and (as shown in formula(II)) R₁₂ is alkyl, e.g., methyl. In some embodiments, Y and Y′ togetherrepresent oxygen; and X represents a substituted amine or a groupA-(CH₂)_(m)—R₁₃, wherein A represents O or N, R₁₃ is a saturated orunsaturated, substituted or unsubstituted carbocyclic or heterocyclicgroup containing at least one hetero atom which is nitrogen, oxygen, orsulfur, and wherein the substituents are independently selected fromalkyl, alkoxy, alkylmercapto, alkylsulfoxide, alkylsulphone, cyano,halogen, hydroxy, amino alkyamino, dialkylamino, azido, and when R₁₃represents substituted or unsubstituted aryl, the aryl substituents maybe alkyl, alkyloxy, alkylmercapto, amino, hydroxyl or halogen; andwherein m is 0, 1 or 2.

In some embodiments, Z represents carbon. In some embodiments, m is 0or 1. In some embodiments, the carbocyclic ring is optionallysubstituted phenyl. In some embodiments, the heterocyclic ring is anoptionally substituted, saturated or unsaturated 5/6-membered ringcontaining at least one S, N or O, e.g., pyridine, piperidine,piperazine, morpholine, pyrrole, pyrrolidine, imidazole, oxazole, and/orthiazole.

In some embodiments, the compounds of the present invention arerepresented by formula

-   wherein R, R₀, R₁ and R₄ are each hydrogen;-   R₂ and R₃ are selected from hydrogen, lower alkoxy, substituted or    unsubstituted aryl(alkyl)oxy, provided that at least one of R₂ and    R₃ is lower alkoxy or substituted or unsubstitutedaryl(alkyl)oxy,    and in some embodiments, R₂ and R₃ are lower alkoxy or benzyloxy,    and in some embodiments R₂ and R₃ are each lower alkoxy;-   R₅ is not present, and R₆ and R₁₀ each represent hydrogen;-   at least one of R₇, R₈ and R₉ is lower alkoxy or substituted or    unsubstituted aryl(alkyl)oxy, and in some embodiments, at least two    of R₇, R₈ and R₉ are lower alkoxy or substituted or unsubstituted    aryl(alky)oxy, and in some embodiments, two of R₇, R₈ and R₉ are    lower alkoxy and one of R₇, R₈ and R₉ is hydrogen, halogen (e.g., F)    or amino or (di)alkylamino, and in some embodiments R₇ and R₉ are    lower alkoxy, and R₈ is lower alkoxy, wherein the substituents are    defined as per formula (I), and in some embodiments, each of R₇, R₈    and R₉ are lower alkoxy. In some embodiments, at least one of R₂,    R₃, R₇, R₈ and R₉ is substituted or unsubstituted aryl(alkyl)oxy,    e.g., benzyoxy, wherein the benzyloxy substituents include alkoxy    (e.g., methoxy), alkyl, alkylamino, dialkylamino, acylamino, and    halo;-   R₁₁ represents hydrogen;-   R₁₂ represents methyl; and-   X represents hydroxyl or (as defined in formula (I)),    -A-(CH₂)_(m)—R₁₃, wherein in some embodiments, A is O and m is 1.

Compounds of formula (I) and (II) can have one or more asymmetriccarbons and thus such compounds are capable of existing as enantiomersor diastereomers. Unless otherwise specified, the present inventionincludes such enantiomers or diastereomers, including any racematesthereof. If desired, the separate enantiomers or diastereomers can besynthesized from appropriate chiral starting materials, or the racematescan be resolved by conventional procedures, which are well-known tothose skilled in the art, such as chiral chromatography, fractionalcrystallization of diastereomers or diastereomeric salts, and the like.Certain compounds can exist as geometrical isomers, such as, forexample, compounds with double-bonded substituents with geometricalisomers Z and E, and the present invention includes all such isomers,including certain isomers, for example, the Z isomers.

In some embodiments, the compound of formula (I) or (II) is in the formof a pharmaceutically acceptable salt. In some embodiments, thepharmaceutically acceptable salt is a sodium salt, a hydrochloride saltor a citric acid salt. In some embodiments, e.g., when Y, Y′ and X forman ester group, the compound of formula (I) or (II) may be said to be inthe form of a prodrug wherein the ester group is cleaved followingadministration.

In some embodiments, the compound is(Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)aceticacid (“compound 9”), or a pharmaceutically

acceptable salt or prodrug thereof (wherein in the latter, the hydroxylgroup is replaced by a substituted amine or a group A-(CH₂)_(m)—R₁₃ (asdefined above).

In some other embodiments, compounds of formulas (I) and (II) (includingprodrugs thereof) are as follows:

Structure IUPAC names

(Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)acetic acid

(Z)-2-(1-(4-(benzyloxy)benzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetic acid

(Z)-2-(1-(3-fluoro-4,5- dimethoxybenzylidene)-5,6-dimethoxy-2-methyl-1H-inden-3-yl)acetic acid

(Z)-2-(1-(4-(dimethylamino)-3,5- dimethoxybenzylidene)-5,6-dimethoxy-2-methyl-1H-inden-3-yl)acetic acid

(Z)-2-(1-(4-(benzyloxy)benzylidene)-5,6-dimethoxy-2-methyl-1H-inden-3-yl)acetic acid

(Z)-2-(1-(4-(benzyloxy)-3,5- dimethoxybenzylidene)-5,6-dimethoxy-2-methyl-1H-inden-3-yl)acetic acid

(Z)-2-(1-(4-(benzyloxy)-3,5- dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetic acid

(Z)-2-(1-(4-(benzyloxy)-3- methoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetic acid

(Z)-2-(1-(4-(benzyloxy)-3-fluoro-5-methoxybenzylidene)-5-methoxy-2-methyl- 1H-inden-3-yl)acetic acid

(Z)-2-(1-(4-(benzyloxy)-3-chloro-5-methoxybenzylidene)-5-methoxy-2-methyl- 1H-inden-3-yl)acetic acid

(Z)-2-(1-(3,5-dimethoxy-4-((4- methoxybenzyl)oxy)benzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetic acid

(Z)-2-(1-(4-((3,4-dimethoxybenzyl)oxy)-3,5-dimethoxybenzylidene)-5,6-dimethoxy-2- methyl-1H-inden-3-yl)acetic acid

(Z)-2-(1-(3,5-dimethoxy-4-((4- methoxybenzyl)oxy)benzylidene)-5,6-dimethoxy-2-methyl-1H-inden-3-yl)acetic acid

2-(dimethylamino)ethyl (Z)-2-(5,6- dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3- yl)acetate

2-(dimethylamino)ethyl (Z)-2-(1-(4- (benzyloxy)benzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetate

2-(4-methylpiperazin-1-yl)ethyl (Z)-2-(5,6- dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3- yl)acetate

2-(4-methylpiperazin-1-yl)ethyl (Z)-2-(1-(4-(benzyloxy)benzylidene)-5-methoxy-2- methyl-1H-inden-3-yl)acetate

2-(piperidin-1-yl)ethyl (Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)- 1H-inden-3-yl)acetate

2-morpholinoethyl (Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H- inden-3-yl)acetate

(1-methylpyrrolidin-2-yl)methyl (Z)-2-(5,6- dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3- yl)acetate

1-methylpyrrolidin-3-yl (Z)-2-(5,6- dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3- yl)acetate

(1-methylpyrrolidin-3-yl)methyl (Z)-2-(5,6- dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3- yl)acetate

1-methylpiperidin-4-yl (Z)-2-(5,6- dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3- yl)acetate

(1-methylpiperidin-2-yl)methyl (Z)-2-(5,6- dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3- yl)acetate

(1-methylpiperidin-2-yl)methyl (Z)-2-(1-(4-(benzyloxy)-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetate

(1,4-dimethylpiperazin-2-yl)methyl (Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5- trimethoxybenzylidene)-1H-inden-3-yl)acetate

(1,4-dimethylpiperazin-2-yl)methyl (Z)-2-(1-(4-(benzyloxy)-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetate

(Z)-2-(1-(4-((4-(dimethylamino)benzyl)oxy)-3,5-dimethoxybenzylidene)-5,6-dimethoxy- 2-methyl-1H-inden-3-yl)aceticacid

(Z)-2-(1-(4-(benzo[d][1,3]dioxol-5-ylmethoxy)-3,5-dimethoxybenzylidene)-5,6-dimethoxy-2-methyl-1H-inden-3-yl)acetic acid

(Z)-2-(1-(3,5-dimethoxy-4-((3,4,5-trimethoxybenzyl)oxy)benzylidene)-5,6-dimethoxy-2-methyl-1H-inden-3-yl)acetic acidIII. Methods of Making the Inventive Compounds

In general, the compounds of formula (I) and (II) can be synthesized inaccordance with the following reaction schemes.

Wherein: W represents R₁, R₂, R₃ and R₅.

A substituted benzaldehyde may be refluxed with propionic anhydride inthe presence of sodium propionate. The resulting solid may becatalytically hydrogenated at 50° C. to obtain the substituted2,3-dihydro-cinnamic acid. The acid may be treated with polyphosphoricacid (PPA) to afford a cyclic ketone. Treatment of the ketone with2-cyanoacetic acid, followed by reaction with potassium hydroxide, willyield the substituted idenyl-acetic acid. Reaction of the acid with asubstituted benzaldehyde in the presence of sodium methoxide in methanolwill afford the benzylidene-indenylacetic acid. Treatment of the acidwith carbonyl diimidazole (CDI) followed by adding a primary alcohol oran amine will yield the ester and amide. See, also e.g., U.S. Pat. No.6,603,818.

IV. Pharmaceutical Compositions

In another aspect of the present invention, a compound of formula (I) or(II), or a pharmaceutically acceptable salt thereof, can be formulatedinto a composition, e.g., a pharmaceutical formulation or composition.In that respect, the present invention further provides a compositionthat includes an effective amount of compound of formula (I) or (II),which may be in the form of a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier. The effective amount caninclude an amount that produces a protective, therapeutic orprophylactic response in a subject to whom a compound or composition ofthe present invention is administered.

Any suitable pharmacologically or physiologically acceptable carrier canbe utilized; including for example, vehicles, solvents, adjuvants,excipients and diluents. One skilled in the art can easily determine thetype of administration for the exact formulation or the compositionbased on the intended route of administration.

By way of example only, in the case of oral preparations, a compound offormulas (I) and (II) can be combined, if desired, with appropriateadditives to make tablets, powders, granules, capsules, liquids, gels,syrups, slurries, solutions and suspensions.

Suitable additives for solid formulations such as these may include forexample, lactose, mannitol, corn starch and potato starch. Suitableadditives also can include binders, for example crystalline cellulose,cellulose derivatives, acacia, or gelatins; disintegrants, for example,corn starch, potato starch and sodium carboxymethylcellulose; andlubricants such as talc and magnesium stearate. Further additives suchas, for example, diluents, buffering agents, moistening agents,preservatives, and/or flavoring agents, and the like, can be included inthe compositions.

The compounds of formulas (I) and (II) can also be made into an aerosolformulation for administration by inhalation. Such aerosol formulationscan be placed into pressurized acceptable propellants such asdichlorodifluoromethane, propane and nitrogen.

The compounds of formulas (I) and (II) can be formulated intosuppositories by admixture with a variety of bases such as emulsifyingbases or water-soluble bases. The suppository formulations can beadministered rectally, and can include vehicles such as cocoa butter,carbowaxes, and polyethylene glycols, which melt at body temperature butare solid at room temperature.

In some embodiments, the compound of formula (I) or (II) may beformulated for ocular administration, such as an eye drop or eyeointment.

The compound of formula (I) or (II) may be added to a base solvent togive an aqueous solution or suspension and then the pH is adjustedwithin a range from 4 to 10, e.g., from 5 to 9. The eye drop may besubjected to a sterilization treatment so as to obtain a sterileproduct, and the sterilization treatment can be carried out in any stageof the production process. The concentration of the compound of formula(I) or (II) in the eye drop may be within a range from about 0.001 toabout 5% (W/V), e.g., about 0.5 to about 1.0% (W/V). The dose may varydepending on the degree of symptoms and constitution of patients and theeye drop may be applied 1 to 4 times per day in an amount of severaldrops. This dose is merely a measure and can deviate from the aboverange according to the pathological conditions of the treatment.

To the eye drop, various additives such as buffering agents, isotonizingagents, antiseptics, pH adjustors, thickeners, chelating agents and,solubilizing agents may be appropriately added. Examples of thebuffering agent include citrate buffering agent, tartaric acid bufferingagent, acetate buffering agent and amino acid. Examples of theisotonizing agent include saccharides such as sorbitol, glucose andmannitol, polyhydric alcohols such as glycerin, polyethylene glycol andpropylene glycol, and salts such as sodium chloride. Examples of theantiseptic include paraoxybenzoate esters such as methyl paraoxybenzoateand ethyl paraoxybenzote, benzyl alcohol, phenethyl alcohol, sorbic acidor salts thereof. Examples of the pH adjustor include phosphoric acidand sodium hydroxide. Examples of the thickener include hydroxyethylcellulose, hydroxypropyl cellulose, methyl cellulose,hydroxypropylmethyl cellulose, carboxymethyl cellulose and saltsthereof. Examples of the chelating agent include sodium edetate, sodiumcitrate and condensed sodium phosphate, and examples of the solubilizingagent include ethanol and polyoxyethylene hardened castor oil.

The eye ointment may be obtained by mixing the compound of formula (I)or (II) with an eye ointment base such as purified lanolin, whitepetrolatum, macrogol, plastibase or liquid paraffin, and is preferablysubjected to a sterilization treatment so as to obtain a sterileproduct. The concentration of the phenylazole compounds of the presentinvention in the eye ointment is within a range from about 0.0001 toabout 5% (W/V), and preferably from 0.5 to 1% (W/V). The dose variesdepending on the degree of symptoms and constitution of patients and theeye ointment may be applied 1 to 4 times per day. This dose is merely ameasure and can deviate from the above range according to thepathological conditions of the treatment.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site where treatment is required, such as liniments, lotions creams,ointments or pastes, and drops.

Lotions of the present invention including those suitable forapplication to the skin or eye. An eye lotion may include a sterileaqueous solution, optionally containing a bactericide, and may beprepared by methods similar to those for the preparation of drops.Lotions or liniments for application to the skin may also include anagent to hasten drying and to cool the skin, such as an alcohol oracetone, and/or a moisturizer such as glycerol or an oil such as castoroil or arachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy basis. The basis may include hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives, or a fattyacid such as stearic or oleic acid together with an alcohol such aspropylene glycol or macrogels. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurface active such as sorbitan esters or polyoxyethylene derivativesthereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

For parental administration, the compositions are preferably formulatedin a sterilized pyrogen-free form. For injection, the agents of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiological saline buffer. Additionally, suspensions ofthe active compounds may be prepared as appropriate oily injections ofthe active compounds may be prepared as appropriate oily injectionsuspensions. Suitable lipophilic solvents or vehicles include fatty oilssuch as sesame oil, synthetic fatty acid esters, such as ethyl oleate ortriglycerides, and liposomes. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension, such assodium carbomethyl cellulose, sorbitol, and dextran. Optionally, thesuspension may also contain suitable stabilizers or agents whichincrease the solubility of the compounds to allow for the preparation ofhighly concentrated solutions.

The determination of the appropriate dose of a compound of formula (I)or (II) is well within the ordinary skill of the art, as areoptimization of dosage and the formulation type for a given subject.Thus, for example, as described herein below, the compounds can beformulated for administration via any standard or medically acceptedroute. The effective amount and method of administration of compoundswill vary based upon the sex, age, weight and disease stage of thesubject, whether the administration is therapeutic or prophylactic, andother factors apparent to those skilled in the art. Those skilled in theart will derive appropriate dosages and schedules of administration tosuit the specific circumstances and needs of the subject, e.g.,dependent on the subject's weight, the severity of the affliction, themanner of administration and the judgment of the prescribing physician.

Although the exact dosage will be determined on a drug-by-drug basis, inmost cases, some generalizations regarding the dosage can be made. Thedaily dosage regimen for an adult human patient may be, for example, adose of between 1 mg and 500 mg, e.g., between about 1 mg and about 250mg, e.g., about 150 to about 200 mg. In some embodiments, the oraldosage form is about 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 125 mg, 150 mg,175 mg, 200 mg, 225 mg, 250 mg or 300 mg.

The term “unit dosage form” as used herein refers to physically discreteunits suitable as unitary dosages for human and animal subjects, eachunit containing a predetermined quantity of at least one compound offormula (I) or (II). The unit dosage can be determined by methods knownto those of skill in the art, for example, by calculating the amount ofactive ingredient sufficient to produce the desired effect inassociation with a pharmaceutically acceptable carrier. Thespecifications for the unit dosage forms that can be used in accordancewith the present invention depend on the particular effect to beachieved and the particular pharmacodynamics associated with thecompound(s) in the individual subject.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack or dispenser device may be accompanied byinstructions for administration.

V. Methods of Use

Normal metabolism produces free radical molecules. Free radicals areatoms or molecules which have at least one unpaired electron in theouter orbital. These radicals are the same as generated by externalradiation and include hydrogen peroxide and superoxide. Mitochondria arethe main source of oxygen free radicals under normal conditions. Freeradicals can react with any biological molecule (proteins, lipids,sugars, DNA) altering its structure and often also its function.Therefore living organisms are provided with a rich system ofantioxidant defenses whose main purpose is to prevent the free radicalsattack to other molecules. Antioxidants can also be supplied in the formof nutrition (e.g., phytochemicals in fruit and vegetables). Oxidativestress arises from an imbalance of these radicals and antioxidants as aresult of which unneutralized radicals damage DNA and othermacromolecules. Oxidative stress occurs to some extent in everyone.However, levels of oxidative stress and ROS that substantially exceedthe mean plus a standard deviation (relative to a population ofdisease-free individuals) can be recognized as a cause of a multitude ofdiseases, may make the body more susceptible to other disease initiatingfactors, inhibit endogenous defenses and repair processes, and enhancedisease progression.

In general, the compounds and compositions of the present invention maybe used to protect cells including normal cells against oxidativedamage. Such uses or methods include: preventing or inhibitingischemic/reoxygenation injury in a patient, particularly in themyocardium and central nervous system; preserving organs for transplantin an anoxic, hypoxic, or hyperoxic state prior to transplant;protecting normal tissues from free radical-induced damage consequent toexposure to ionizing radiation and/or chemotherapy; protecting cells andtissues from free radical-induced injury consequent to exposure toxenobiotic compounds which form free radicals; enhancingcryopreservation of cells, tissues, organs, and organisms by increasingviability of recovered specimens; prophylactic administration to preventcellular senescence, cataract formation, formation of malondialdehydeadducts, HIV pathology and macromolecular crosslinking, such as collagencrosslinking; protection of normal cells from damage caused byenvironmental factors e.g., rays from the sun, such as for example,ultra violet rays and gamma rays, and carcinogens; protection of normalcells in a subject from damage caused by aging, including age-relatedimmune deficiency and premature aging disorders, cancer, cardiovasculardisease, cerebrovascular disease, radiation injury, alcohol-mediateddamage (including Wernicke-Korsakoff s syndrome), inflammatory andauto-immune disease, drug toxicity, amyloid disease, overload syndromes(iron, copper, etc.), multi-system organ failure, andendotoxemia/sepsis; and enhancing wound recovery.

Thus, in some embodiments, a compound of formula (I) or (II) isadministered to a subject undergoing or expected to undergo: an ischemicepisode, such as a myocardial infarction, cerebral ischemic event,transplantation operation, open heart surgery, elective angioplasty,coronary artery bypass surgery, brain surgery, renal infarction,traumatic hemorrhage, and tourniquet application; antineoplastic orantihelminthic chemotherapy employing a chemotherapeutic agent whichgenerates free radicals; endotoxic shock or sepsis; exposure to ionizingradiation; exposure to exogenous chemical compounds which are freeradicals or produce free radicals; thermal or chemical burns orulcerations; hyperbaric oxygen; and apoptosis of a predetermined cellpopulation (e.g., lymphocyte apoptosis).

In some embodiments, the present invention provides methods for theprevention, inhibition, amelioration, or treatment of a non-cancerousdisease or condition associated with oxidative stress (and related toexcess ROS production) in a subject by administration of aprophylactically therapeutically effective amount of a compound offormula (I) or (II) to the subject. Examples of conditions associatedwith oxidative stress include reperfusion injury, wound healing, toxichepatitis, viral hepatitis, chronic organ disease (e.g., chronic lungdisease, chronic obstructive pulmonary disease, chronic viral hepatitis,chronic renal disease, chronic pancreatitis, chronic prostatitis,chronic inherited bleeding disorders (e.g., hemophilia, von Willebranddisease), and chronic bone disease (e.g., osteogenesis imperfect,Paget's disease), oxidative stress from dialysis, renal toxicity, kidneyfailure, ulcerative colitis, bacterial infection, viral infections,upper respiratory tract diseases, oxidative stress due to sun damage,eczema, atopic dermatitis, polymyositis, dermatitis, herpetiformis, andpre-cancerous conditions.

Certain of the conditions characterized by oxidative stress fall withinthe cardiovascular group, including myocardial ischemia, myocardialinfarction, cardiopulmonary inflammatory disorders; and heart failure(including chronic and congestive heart failure).

Another group of conditions characterized by oxidative stress fallwithin the cerebrovascular and neurologic group, including stroke,cerebral ischemia, retinal ischemia, post-surgical cognitivedysfunctions (e.g., following bypass surgery), peripheral neuropathyspinal cord injury, head injury and surgical trauma, andneurodegenerative disorders including Alzheimer's, dementia andParkinson's disease.

In some embodiments, a compound of formula (I) or (II) is used to treatischemia. Ischemia refers to a disorder caused by an imbalance betweensupply and demand of oxygen to tissue, usually caused by a reduction inblood flow to the tissue. Organs such as the heart and brain are mostvulnerable to ischemia due to their high extraction of oxygen.Reperfusion is the process of restoring blood flow to the tissue.Ischemia and reperfusion result in different damage to the tissuesdeprived of oxygen. The reduction of blood flow decreases the productionof high energy phosphates. During ischemia, cells are damaged and theirmitochondria becomes less efficient. Restarting blood flow after morethan about ten minutes of ischemia is typically more damaging than theischemia itself because the damaged cells produce large amounts ofreactive oxygen species.

Subjects at risk of ischemia include those having previously had heartdisease, those having elevated biochemical markers of the disease (e.g.,protein C), those identified as having blockage of blood vessels byangioplasty or MRI imaging, and those undergoing a surgical procedurerequiring temporary obstruction of blood vessels.

In some embodiments, a compound of formula (I) or (II) is used to treatstroke. Stroke is a sudden loss of brain function resulting frominterference with the blood supply to the central nervous system. Acutestroke can be classified either as ischemic (80% of stroke cases), whichcan be further classified to extra-cranial embolism and intracranialthrombosis, or a hemorrhagic stroke (20% of stroke cases), which can befurther classified to intracerebral hemorrhage and subarachnoidhemorrhage. Ischemic stroke accounts for 70 to 80% of all strokes andhemorrhagic stroke accounts for the remainder. Free radicals play animportant role in the pathogenesis of stroke. In most cases, subjects atrisk of stroke can be determined by presence of one, and usually atleast two of the following risk factors: high blood pressure, heartdisease, high cholesterol levels, sleep apnea, previous occurrence ofstroke, smoking, excessive alcohol consumption and excessive weight.

Another group of diseases characterized by oxidative stress andinvolving inflammatory and/or autoimmune components includes diabetes;renal disease; pre-menstrual syndrome; asthma; rheumatoid arthritis;osteoarthritis, muscle fatigue; irritable bowel syndrome, inflammatorybowel disease and intermittent claudication.

Another group of diseases characterized by oxidative stress fall withinthe group of dermatologic conditions, including, prevention andprotecting skin tissue against age-related damage or damage resultingfrom insults such as harmful ultraviolet (UV) radiation, stress andfatigue.

In yet further embodiments, various retinal diseases characterized byoxidative stress may be treated with the compounds of formula (I) or(II).

Photoreceptor outer segments contain rhodopsin as well as the highestcontent of DHA of any cell type. In contact with the photoreceptor tipsis a monolayer of cells, the retinal pigment epithelium (RPE), derivedfrom neuroepithelium. These cells are the most active phagocytes of thebody. In a daily cycle, they engulf and phagocytize the distal tips ofphotoreceptor outer segments, thereby participating in rod outer segmentrenewal in a process that is balanced by addition of new membrane to thebase of the outer segments. The conservation of DHA in photoreceptors issupported by retrieval through the interphotoreceptor matrix, whichsupplies the fatty acid for the biogenesis of outer segments. See,Stinson, et al., J. Lipid Res. 32:2009-2017 (1991).

RPE cells also perform several other functions, including transport andreisomerization of bleached visual pigments, and contribute to themaintenance of the integrity of the blood-outer retinal barrier. Retinaldetachment or trauma triggers dysfunctions in the RPE cells that lead tothe onset and development of proliferative vitreoretinopathy.

RPE cells are essential for photoreceptor cell survival. When RPE cellsare damaged or die, photoreceptor function is impaired, and the cellsdie as a consequence. Thus, oxidative stress-mediated injury and celldeath in RPE cells impair vision, particularly when the RPE cells of themacula are affected. The macula is responsible for visual acuity.

In summary, retinal photoreceptors are packed with mitochondria and haveextremely high metabolic activity and oxygen consumption. Sinceproduction of ROS the electron transport chain is a major source ofoxidative stress, photoreceptors are challenged under normalcircumstances. In patients with retinitis pigmentosa (RP), one of anumber of different mutations causes death of rods which drasticallyreduces oxygen consumption and elevates oxygen levels in the outerretina. Prolonged exposure to high levels of oxygen causes progressiveoxidative damage to cones, Shen et al., J. Cell Physiol. 203:457-464(2005), and their gradual death results in progressive constriction ofvisual fields and eventual blindness.

Thus, in some embodiments, a compound of formula (I) or (II) is used totreat retinitis pigmentosa (RP). RP is a type of progressive retinaldystrophy, a group of inherited disorders in which abnormalities of thephotoreceptors (rods and cones) or the retinal pigment epithelium (RPE)of the retina lead to progressive visual loss. Affected individualsfirst experience defective dark adaptation or nyctalopia (nightblindness), followed by reduction of the peripheral visual field (knownas tunnel vision) and, sometimes, loss of central vision late in thecourse of the disease.

The diagnosis of retinitis pigmentosa relies upon documentation ofprogressive loss in photoreceptor function by electroretinography (ERG)and visual field testing. The mode of inheritance of RP is determined byfamily history. At least 35 different genes or loci are known to cause“nonsyndromic RP” (RP that is not the result of another disease or partof a wider syndrome). RP is commonly caused by a mutation in the opsingene, but can be caused by mutations in a number of other genesexpressed systemically or exclusively in the eye involved in the visualcycle.

Oxidative damage has also been implicated in another highly prevalenteye disease, age-related macular degeneration (AMD). Thus, in someembodiments, a compound of formula (I) or (II) is used to treat AMD.

Yet other ocular diseases and conditions that may be treated withcompounds of the present invention include both wet and dry, diabeticretinopathy, Lebers optic neuropathy, and optic neuritis.

Compounds of formula (I) or (II) may also be useful in the treatment ofsubjects who are at risk or predisposed to cancer. These subjects thatwould be the recipients of such treatment have pre-cancerous conditions,which are known in the art as a group of disorders that have a malignantpredisposition. Representative examples of pre-cancerous conditions thatmay be treated in accordance with the present inventive compoundsinclude adenomas/polyps in subjects with familial adenomatous polyposis(Gardner's syndrome), sporadic adenomatous polyposis, or precancerousconditions associated with the hereditary non-polyposis colon cancer(Lynch syndrome), inflammatory bowel disease, or Crohn' s disease.Examples of precancerous conditions of other tissues include cervicaldysplasia or squamous intraepithelial lesion (e.g., diagnosed by a papsmear), prostatic intraepithelial neoplasia (PIN), superficial bladdercancer, also known as transitional cell carcinoma in situ, precancerouslesions of the breast, precancerous lesions of the lungs, actinickeratosis, Barrett's esophagus, precancerous melanoma moles,precancerous conditions of the uterus/vulva, precancerous conditions ofthe ovary, atrophic gastritis, precancerous conditions of the oralcavity, general dysplastic conditions, squamous metaplasia,intraepithelial neoplasia, and precancerous conditions in the head orneck.

The compounds of the present invention can, for example, be administeredby any medically accepted route, including, for example, ophthalmically(e.g., intraocularly, intravitreally, subretinally), parenterally (e.g.,intravenously, intraarterially, subdermally, intraperitoneally,intramuscularly, or subcutaneously) or orally, topically, buccally,nasally, transmucosally, or directly to a diseased organ, e.g.,surgically or by catheter.

Compositions of the invention can also be administered in vitro to acell (for example, to prevent oxidative damage during ex vivo cellmanipulation, for example of organs used for organ transplantation or inin vitro assays) by simply adding the composition to the fluid in whichthe cell is contained.

It will be appreciated that an agent (e.g.,(Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)aceticacid, or a pharmaceutically acceptable salt or prodrug thereof) orcomposition, as described herein, can be administered in combinationwith one or more additional pharmaceutical agents (e.g., therapeuticallyand/or prophylactically active agents), which are different from theagent or composition and may be useful as, e.g., combination therapies.The agents or compositions can be administered in combination withadditional pharmaceutical agents that improve their activity (e.g.,activity (e.g., potency and/or efficacy) in treating a disease in asubject in need thereof, in preventing a disease and/or injuriousincident in a subject in need thereof, in reducing the risk ofdeveloping a disease in a subject in need thereof, etc. in a subject orcell. In certain embodiments, a pharmaceutical composition describedherein including an agent (e.g.,(Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)aceticacid, or a pharmaceutically acceptable salt or prodrug thereof)described herein and an additional pharmaceutical agent shows asynergistic effect that is absent in a pharmaceutical compositionincluding one of the agent and the additional pharmaceutical agent, butnot both.

In some embodiments of the disclosure, a therapeutic or prophylacticagent distinct from a first therapeutic or prophylactic agent of thedisclosure is administered prior to, in combination with, at the sametime (i.e., co-administered), or after administration of the agent ofthe disclosure. In some embodiments, the second therapeutic agent isselected from the group consisting of aspirin, a blood pressuremedication, a type II diabetes medication, an mTOR inhibitor and/orother chemotherapeutic agent, etc.

The agent or composition can be administered concurrently with (i.e.,co-administered), prior to, or subsequent to one or more additionalpharmaceutical agents, which may be useful as, e.g., combinationtherapies. Pharmaceutical agents include therapeutically active agents.Pharmaceutical agents also include prophylactically active agents.Pharmaceutical agents include small organic molecules such as drugcompounds (e.g., compounds approved for human or veterinary use by theU.S. Food and Drug Administration as provided in the Code of FederalRegulations (CFR)), peptides, proteins, carbohydrates, monosaccharides,oligosaccharides, polysaccharides, nucleoproteins, mucoproteins,lipoproteins, synthetic polypeptides or proteins, small molecules linkedto proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs,nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides,lipids, hormones, vitamins, and cells. In certain embodiments, theadditional pharmaceutical agent is a pharmaceutical agent useful fortreating and/or preventing a disease described herein. Each additionalpharmaceutical agent may be administered at a dose and/or on a timeschedule determined for that pharmaceutical agent. The additionalpharmaceutical agents may also be administered together with each otherand/or with the agent or composition described herein in a single doseor administered separately in different doses. The particularcombination to employ in a regimen will take into account compatibilityof the agent described herein with the additional pharmaceuticalagent(s) and/or the desired therapeutic and/or prophylactic effect to beachieved. In general, it is expected that the additional pharmaceuticalagent(s) in combination be utilized at levels that do not exceed thelevels at which they are utilized individually. In some embodiments, thelevels utilized in combination will be lower than those utilizedindividually.

The additional pharmaceutical agents include, but are not limited to,aspirin, blood pressure medications, type II diabetes medications, mTORinhibitors, other anti-cancer agents, immunomodulatory agents,anti-proliferative agents, cytotoxic agents, anti-angiogenesis agents,anti-inflammatory agents, immunosuppressants, anti-bacterial agents,anti-viral agents, cardiovascular agents, cholesterol-lowering agents,anti-diabetic agents, anti-allergic agents, contraceptive agents, andpain-relieving agents. In certain embodiments, the agents describedherein or pharmaceutical compositions can be administered in combinationwith an anti-cancer therapy including, but not limited to, surgery,radiation therapy, transplantation (e.g., stem cell transplantation,bone marrow transplantation), immunotherapy, and chemotherapy.

Dosages for a particular agent of the instant disclosure may bedetermined empirically in individuals Who have been given one or moreadministrations of the agent.

Administration of an agent of the present disclosure can be continuousor intermittent, depending, for example, on the recipient'sphysiological condition, whether the purpose of the administration istherapeutic or prophylactic, and other factors known to skilledpractitioners. The administration of an agent may be essentiallycontinuous over a preselected period of time or may be in a series ofspaced doses.

Guidance regarding particular dosages and methods of delivery isprovided in the literature; see, for example, U.S. Pat. Nos. 4,657,760;5,206,344; or 5,225,212. It is within the scope of the instantdisclosure that different formulations will be effective for differenttreatments and different disorders, and that administration intended totreat a specific organ or tissue may necessitate delivery in a mannerdifferent from that to another organ or tissue. Moreover, dosages may beadministered by one or more separate administrations, or by continuousinfusion. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of disease symptoms occurs. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques and assays.

VI. Kits

The instant disclosure also provides kits containing agents of thisdisclosure for use in the methods of the present disclosure. Kits of theinstant disclosure may include one or more containers comprising apurified agent (e.g.,(Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)aceticacid, or a pharmaceutically acceptable salt or prodrug thereof) of thisdisclosure and/or may contain agents for identifying a subject atelevated risk for an ischemic event. In some embodiments, the kitsfurther include instructions for use in accordance with the methods ofthis disclosure. In some embodiments, these instructions comprise adescription of administration of the agent to treat, prevent and/ordiagnose, e.g., a risk factor that predisposes a subject to an ischemicevent, according to any of the methods of this disclosure. In someembodiments, the instructions comprise a description of how to detect asubject at elevated risk of an ischemic event of a tissue, optionally ofthe heart, brain, kidneys, liver or lung. The kit may further comprise adescription of selecting an individual suitable for treatment based onidentifying whether that subject has an ischemia-predisposing riskfactor(s).

The instructions generally include information as to dosage, dosingschedule, and route of administration for the intended treatment. Thecontainers may be unit doses, hulk packages (e.g., multi-dose packages)or subunit doses. Instructions supplied in the kits of the instantdisclosure are typically written instructions on a label or packageinsert (e.g., a paper sheet included in the kit), but machine-readableinstructions (e.g., instructions carried on a magnetic or opticalstorage disk) are also acceptable.

The label or package insert indicates that the composition is used fortreating or preventing, e.g., an ischemic event and/or the negativeeffects attributable to an ischemic event, in a subject. Instructionsmay be provided for practicing any of the methods described herein.

The kits of this disclosure are in suitable packaging. Suitablepackaging includes, but is not limited to, vials, bottles, jars,flexible packaging (e.g., sealed Mylar or plastic bags), and the like.Also contemplated are packages for use in combination with a specificdevice, such as an inhaler, nasal administration device (e.g., anatomizer) or an infusion device such as a minipump. A kit may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The container may also have a sterile access port(e.g., the container may be an intravenous solution bag or a vial havinga stopper pierceable by a hypodermic injection needle). In certainembodiments, at least one active agent in the composition is(Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)aceticacid, or a pharmaceutically acceptable salt or prodrug thereof. Thecontainer may further comprise a second pharmaceutically active agent.Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

WORKING EXAMPLES

The present invention is now described in terms of the followingnon-limiting examples.

Example 1 Materials and Methods

Chemical synthesis of Compound 9: 3,4-Dimethoxybenzaldehyde was treatedwith propionic anhydride and sodium propionate at 130° C. for 36 h togive (Z)-3-(3,4-dimethoxyphenyl)-2-methylacrylic acid as a colorlesssolid. Catalytic hydrogenation at 40 psi afforded3-(3,4-dimethoxyphenyl)-2-methylpropanoic acid as colorless oil.5,6-Dimethoxy-2-methyl-2,3-dihydro-1H-inden-1-one was obtained bytreating the oil with polyphosphoric acid (PPA) at 50° C. for 20 min,followed by a standard purification procedure; the ketone was refluxedwith 2-cyanoacetic acid, acetic acid and ammonium chloride in toluenefor 36h, followed by hydrolysis with potassium hydroxide in 60% ofethanol to give 2-(5,6-dimethoxy-2-methyl-1H-inden-3-yl)acetic acid.Reaction of the acid with 3,4,5-trimethoxybenzaldehyde and methoxide inmethanol at 85° C. overnight afforded2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzyl)-1H-inden-3-yl)aceticacid (Compound 9) as a yellow solid.

Chemical synthesis of Compound 10: 4-Dimethoxybenzaldehyde was treatedwith propionic anhydride and sodium propionate at 130° C. for 36 h togive (Z)-3-(4-methoxyphenyl)-2-methylacrylic acid as a colorless solid.Catalytic hydrogenation at 40 psi afforded3-(4-methoxyphenyl)-2-methylpropanoic acid as colorless oil.6-Methoxy-2-methyl-2,3-dihydro-1H-inden-1-one was obtained by treatingthe oil with polyphosphoric acid (PPA) at 50° C. for 20 min, followed bya standard purification procedure; the ketone was refluxed with2-cyanoacetic acid, acetic acid and ammonium chloride in toluene for36h, followed by hydrolysis with potassium hydroxide in 60% of ethanolto give 2-(5-methoxy-2-methyl-1H-inden-3-yl)acetic acid. Reaction of theacid with 4-benzyloxy-benzaldehyde and methoxide in methanol at 85° C.overnight afforded(Z)-2-(1-(4-(benzyloxy)benzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)aceticacid (Compound 10) as a yellow oil.

Cultured cells and reagents: Retinal pigmented epithelium cells(ARPE-19) were obtained from American Type Culture Collection (ATCC,Manassas, Va.) and cultured at 37° C. in 5% CO₂. All chemicals werepurchased from Sigma unless specified.

Cell viability assay: Cells were cultured and assayed for cell viabilityessentially as described previously (14). ARPE-19 cells were plated at10,000 cells per well in a 96-well plate. The cells were grown for 18-20hours, the medium discarded in aseptic conditions and replaced withfresh culture medium containing the indicated drug concentration. Theplates were incubated for 24 hours at 37° C. in a 5% CO₂ incubator inthe presence of sulindac or Compound 9. To examine the treatment effectson oxidative damage to RPE cells, cells were washed and incubated inmedia without serum with various concentrations of tert-butylhydroperoxide (TBHP) for an additional 24h. The culture medium was thendiscarded and the cells were thoroughly rinsed in PBS. Cell viabilitywas determined by using the Cell Titer 96 Aqueous One Cell ProliferationAssay (Promega) according to the manufacturer's instructions. The assayutilizes a tetrazolium compound that is converted into a water-solubleformazan by the action of cellular NADH oxidase activity inmetabolically active cells. The formazan was quantified by measuring theabsorbance at 490 nm using a colorimetric microtiter plate reader(SpectraMax Plus; Molecular Devices).

PDE Assay

PDE activity was measured using the IMAP fluorescence polarization PDEassay (Molecular Devices) in which the binding of hydrolyzed fluorescentcyclic nucleotide substrate to the IMAP reagent increases fluorescencepolarization. Each well of a 96-well nonbinding plate contained 0.25mg/mL of recombinant enzyme preparations. Enzymes were incubated withCOMPOUND 9 or vehicle (DMSO) for 30 min at 30° C. before the addition ofa substrate mixture containing 25 nmol/L of fluorescein-cGMP. The DMSOfinal concentration for each experiment was 2%. After 90 min ofincubation at 30° C., the reaction was terminated by the addition ofbinding reagent. The maximum Fluorescence polarization was measuredusing a Synergy4 (BioTek) plate reader.

COX Assay

Compounds were assayed for cyclooxygenase (COX) inhibition using the COXFluorescent Inhibitor Screening Kit from Cayman Chemical (Item #:700100). Briefly, COX enzyme (ovine COX-1 or human recombinant COX-2)diluted in 1× reaction buffer is incubated with the compound or DMSOcontrol for 20 min at 25° C. in a half-area 96-well black microplate.Background wells are also included that contained no enzyme. Added toeach well were heme B, to ensure enzyme activity, and10-acetyl-3,7-dihydroxyphenoxazine (ADHP) assay reagent. After the 20min incubation, arachidonic acid (AA) substrate was added to all wells.As shown by the schematic diagram below, active COX enzyme converts AAto hydroperoxy endoperoxide (PGG₂). Catalyzed by the secondaryperoxidase activity of COX, ADHP can react with PGG₂ to form a highlyfluorescent compound, resorufin. Fluorescence was measured with a530-450 nm excitation wavelength and a 585-595 nm emission wavelength.Decreased fluorescence relative to the DMSO control indicates COXinhibition. See, also, Piazza G A, Keeton A B, Tinsley H N, Gary B D,Whitt J D, Mathew B, Thaiparambil J, Coward L, Gorman, G, Li Y, Sani B,Hobrath J V, Maxuitenko Y Y and Reynolds R C. A Novel SulindacDerivative That Does Not Inhibit Cyclooxygenases, but Inhibits ColonTumor Cell Growth and Induces Apoptosis with Antitumor Activity. CancerPrevention Research 2009; 2: 572-580.

Langendorff Model

The ex vivo Langendorff procedure is a well-established preparation usedto investigate heart physiology, ischemia/reperfusion injury, and othercardiovascular insults (23, 24; also see, e.g., reference 22). SpragueDawley rats (275-325g) were administered either no drug or Compound 9daily (at 0.7 mg/kg) for a total of two days (two administrations intotal). Forty-eight hours after the start of drug exposure, hearts wereremoved and exposed to 45 min no flow ischemia and 2 h reperfusion inthe Langendorff model (hearts were first equilibrated in KHB buffer for10 min and then subjected to 45 min ischemia followed by 2 h reperfusionwith KHB buffer), in the absence of drug.

Cell Death and Viability Assays

In LDH and TTC assays for the Langendorff Preparation, coronary effluentsamples (500 μL) from the Langendorff preparation were obtained uponattachment of excised heart, every 15 min before ischemia, immediatelyafter 45 min ischemia, and at 15 min intervals during reperfusion. LDHwas measured (using a Cytotox-96 nonradioactive cytotoxicity assay kit(Promega), with the absorbance read at 490 nm). Immediately uponcompletion, the heart was sliced into 2-mm cross-sectional pieces andslices were incubated for 30 min with 1% 2,3,5-triphenyl tetrazoliumchloride (TTC) stain in Krebs-Henseleit buffer (KHB) (pH=7.4) at 37° C.to distinguish between viable (red) and nonviable (white) tissue. Tissueslices were stored overnight in 10% formaldehyde before measurement ofinfarct size using NIH-Image J software.

Statistical Analysis

Data were analyzed using two tailed T-test or ANOVA using Graph padprism software. Data are expressed as mean ±standard error (SE) withstatistical significance set at p<0.05.

Example 2 Identification and Initial Evaluation of Indene Derivatives

Sulindac is a known NSAID that has been shown to have anti-canceractivity in experimental rodent models of tumorigenesis and is effectivefor the treatment of precancerous colonic adenomas in patients withfamilial adenomatous polyposis (15-17). However, sulindac, like otherNSAIDs, has gastrointestinal and other toxicities resulting from COXinhibition that may limit their long-term use for cancer chemopreventionor other diseases. To develop safer and more efficacious derivatives,previous studies have focused primarily on synthesizing derivatives thathave reduced COX inhibitory activity and improve potency to inhibittumor cell growth (18, 19). As shown previously, sulindac can alsoprotect normal cells against oxidative damage by initiating a protectivepreconditioning response (9, 10) that is independent of its COXinhibitory activity. Since the protective effect observed with sulindacusing cells in culture required relatively high concentrations of thedrug (10), there has been concern about possible long term toxicityrelated to its COX inhibitory activity.

We initiated a screen of a library of indene derivatives to identifynovel compounds that are: 1) more potent than sulindac in protectingnormal cells against oxidative damage; and 2) have lower orsubstantially lack COX inhibitory activity. In the present studies, aseries of compounds were identified as having the ability to protect RPEcells against oxidative damage using tert-butyl hydroperoxide (TBHP), anorganic peroxide, as an oxidizing agent (10).

The structures and names of the compounds screened, and the assayresults, are shown in the following table.

Cyto-protective Compound Structure activity* IUPAC names 1

inactive sodium (Z)-2-(1-(4- (dimethylamino)benzylidene)-5-fluoro-2-methyl-1H-inden-3-yl)acetate 2

inactive sodium (Z)-2-(1-(4- (dimethylamino)benzylidene)-5-fluoro-2-methyl-1H-inden-3-yl)acetate 3

inactive (Z)-2-(6-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)acetic acid 4

inactive (Z)-2-(5-methoxy-2-methyl-1-(4-((trifluoromethyl)thio)benzylidene)-1H- inden-3-yl)acetic acid 5

inactive (Z)-2-(1-(4-bromo-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl- 1H-inden-3-yl)acetic acid 6

inactive (Z)-2-(1-(4-(dimethylamino)benzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetic acid 7

inactive (Z)-2-(1-(3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetic acid 8

inactive (Z)-2-(2,5-dimethyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)acetic acid 9

active (Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)acetic acid 10

active (Z)-2-(1-(4-(benzyloxy)benzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetic acid 11

inactive (Z)-2-(5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)acetic acid 12

inactive (Z)-2-(5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)acetic acid 13

inactive (Z)-2-(1-(4-hydroxy-3-methoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetic acid 14

inactive (Z)-2-(1-(benzo[d][1,3]dioxol-5-ylmethylene)-5,6-dimethoxy-2-methyl-1H- inden-3-yl)acetic acid 15

inactive (Z)-2-(1-(3-hydroxy-4-methoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetic acid 16

inactive (Z)-2-(1-(3-bromo-4-hydroxy-5-methoxybenzylidene)-5-methoxy-2-methyl- 1H-inden-3-yl)acetic acid 17

inactive (Z)-2-(1-(3,5-dimethoxybenzylidene)-5,6-dimethoxy-2-methyl-1H-inden-3-yl)acetic acid 18

inactive (Z)-N-(furan-2-ylmethyl)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5,6-dimethoxy-2-methyl-1H-inden-3-yl)acetamide 19

inactive (Z)-2-((5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3- yl)methyl)-1H-imidazo[4,5-c]pyridine20

inactive (Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)-N- (furan-2-ylmethyl)acetamide 21

inactive (Z)-2-(1-(3,5-dimethoxybenzylidene)-5,6-dimethoxy-2-methyl-1H-inden-3-yl)-N- (furan-2-ylmethyl)acetamide 22

inactive (Z)-2-(1-(4-(dimethylamino)benzylidene)-5,6-dimethoxy-2-methyl-1H-inden-3-yl)-N- (furan-2-ylmethyl)acetamide 23

inactive (Z)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl- 1H-inden-3-yl)acetic acid*active = more active than Sulindac inactive = less active than Sulindac

As shown in FIG. 2, Compound 9 (25 μM) provided complete protection ofRPE cells from TBHP treatment up to TBHP concentrations of 400 μM,whereas sulindac (200 μM) was no longer protective at TBHPconcentrations above 300 μM. Compound 10 was equally effective assulindac but was more potent, being effective at an 8× lowerconcentration of 25 μM. Compound 9 was found to be effective atconcentrations as low as 20× relative to sulindac at a concentration of10 μM (FIG. 3).

To determine if the cytoprotective activity of Compound 9 might involveCOX-1 and/or COX-2 inhibition, the compound was tested for COXinhibitory activity in enzymatic assays. FIG. 4A shows that with respectto COX-1 inhibition, Compound 9 is least 20× less potent than the knownCOX-1 inhibitor, sulindac sulfide (SS). FIG. 4B shows that with respectto COX-2 inhibition, Compound 9 is approximately 1000× less potent thanthe known COX-2 inhibitor, celecoxib. These results show that Compound 9essentially lacks pharmacologically significant COX-1 or COX-2inhibitory activity.

Both the COX inhibitory sulfide and non-COX inhibitory sulfonemetabolites of sulindac have been reported to inhibit cGMP degradingphosphodiesterases (PDE) with selectivity for PDE isozymes thathydrolyze cGMP. PDE inhibition results in elevated intracellular levelsof cGMP and activation of protein kinase G (PKG). Inhibition of PDE5 andPDE10 inhibition may be responsible for the anticancer activity ofsulindac by a mechanism involving the suppression of cytoplasmic/nuclearlevels of β-catenin and inhibition of the TCF transcription factor,which mediates transcription of multiple proteins, including cell cycleregulatory and pro-survival genes such as cyclin D and survivin (20,21). To determine if the cytoprotective activity of Compound 9 requirescGMP PDE inhibition, the effect of Compound 9 on the enzymatic activityof recombinant PDE isozymes was measured in enzymatic assays.

As shown in FIG. 5, Compound 9 inhibits PDE5 (FIG. 5A) and PDE10 (FIG.5B) with IC50 values of about 32 μM and 7 μM, respectively, which arewithin the same concentration range required for cytoprotection of RPEcells exposed to oxidative damage by THBP. The PDE isozyme selectivityof Compound 9 was also determined by testing the sensitivity of PDEisozymes 1-11 (except PDE6). As shown in FIG. 5C, Compound 9 (25 μM) wasmost active against PDE5 and 10, although there was lower inhibitoryeffects on other cGMP PDE isozymes, including PDE 1, 2, 3 and 11. Theseresults suggest that the elevation of cGMP by Compound 9, leading toincreased activity of PKG, could be responsible for the cytoprotectiveactivity of Compound 9.

We used various metabolic inhibitors to obtain evidence that Compound 9is functioning through a preconditioning mechanism. FIG. 6A shows thattiron, a scavenger of ROS, attenuates the protective effect caused byCompound 9, suggesting a role of ROS in the observed protection. FIG. 6Bshows that chelerythrine, a broad inhibitor of protein kinase C (PKC),significantly reduces the protective effect of Compound 9. Finally, FIG.6C shows that Rp-Br-8-PET-cGMPS, an inhibitor of PKG, reduces theprotection by Compound 9, which is consistent with a role of PDE5 andPDE10 inhibition as shown in FIG. 5. Together, the results in FIG. 6indicate that Compound 9 is very likely initiating a pharmacologicalpreconditioning response.

Example 3 Intraperitoneal (I.P.) Administration of Compound 9 ProvidedSignificant Protection Against Cell Death in a Rat Langendorff Model ofMyocardial Ischemia

The protective (preconditioning) effect of Compound 9 was evaluated inan art-recognized Langendorff model of ischemia/reperfusion injury.Compound 9 was administered via i.p. injection to test animals daily fortwo days, and animal hearts were then removed for performance of theLangendorff procedure (described above and known in the art).

As shown in FIG. 7, after 48 h of Compound 9 treatment prior toischemia/reperfusion injury, levels of LDH released (LDH being a markerof diminished cell viability) were significantly lower after a 45 minperiod of no flow ischemia in the hearts from Compound 9-administeredrats compared to the hearts from animals receiving the no drugadministration (FIG. 7, 45 min ischemia results). The extent ofprotective effect observed for Compound 9 became more demonstrableduring the 2 h reperfusion: Compound 9 markedly protected the heartagainst oxidative damage as seen by the decrease in LDH levels comparedto the no drug control (compare 2 h reperfusion results of FIG. 7).

Infarct size was also significantly reduced in Compound 9-treatedanimals, as compared to control animals. As shown in FIGS. 8A and 8B,TTC staining (in which healthy tissue stains pink/red, whereas damagedtissue appears white) revealed that Compound 9-treated rats exhibitedinfarction sizes below 20%, as contrasted with animals administered nodrug, which exhibited infarction sizes exceeding 50%.

Notably, Compound 9 exhibited enhanced potency of protective effect ascompared to the protective effect previously described for sulindac.Specifically, as shown in FIG. 9, Compound 9 administered via i.p.injection at 0.7 mg/kg demonstrated robust protective effect both duringthe 45 min ischemia phase of the Langendorff procedure and during the 2h reperfusion phase. Meanwhile, sulindac exhibited no protective effectduring the 45 min ischemia phase when administered at 0.7 mg/kg i.p.daily for 48 h prior to the Langendorff procedure, in contrast toprevious results in which a protective effect of sulindac was observedeven during the 45 min ischemia phase, and the protective effect thatwas observed for sulindac during the 2 h reperfusion phase was not asrobust as that observed for Compound 9, when both sulindac and Compound9 were administered at the same dose (0.7 mg/kg) and via the same routeof administration (i.p. injection).

The above studies therefore demonstrated that Compound 9 protected ratintact hearts (Langendorff model) against oxidative damage resultingfrom ischemia/reperfusion. Administration of Compound 9 to rats in vivofollowed by removal of the heart, wash-out of the drug and subsequentischemia and reperfusion resulted in substantial protection againstischemia-induced cell death relative to untreated hearts.

IPC is believed to be an important cellular protective mechanism,especially for organs that have high rates of respiration, such as theheart, brain and retina. There are other agents that have been reportedto protect cells against ischemia/reperfusion damage, including the PDE5inhibitor, sildenafil (Viagra™), and PPAR agonists (20, 21), that mayact like pharmacologically preconditioning agents. Based on our previousstudies (9, 10), and unpublished cell culture results, sulindac hadappeared to be the most effective preconditioning agent, although itsability to inhibit prostaglandin synthesis from suppressingcyclooxygenase posed a significant limitation. In rodent cardiacstudies, sulindac was highly effective in reducing the infarct sizeresulting from ischemic/reperfusion damage at a dose that was less than20% (on a mg per kg basis) of the amount when used in humans as ananti-inflammatory drug (9). However, a more active sulindac derivative,with less or no COX inhibitory activity, would be safer and moreefficacious. Thus, compounds of the present invention, e.g., Compound 9,that may be at least 10 times more active than sulindac (on a molarbasis) in protecting RPE cells and other types of cells from oxidativedamage, and which exhibit no significant COX inhibitory activity, areherein identified as likely to offer surprising and unexpected healthadvantages for clinical use.

All publications cited in the specification, including patentpublications and non-patent publications, are indicative of the level ofskill of those skilled in the art to which this invention pertains. Allthese publications are herein incorporate by reference to the sameextent as if each individual publication were specifically andindividually indicated as being incorporated by reference.

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Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

What is claimed is:
 1. A pharmaceutical composition comprising thecompound (Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-1)acetic acid, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier,wherein the amount of the compound is effective to protect cells againstoxidative damage, and wherein the composition is in a form suitable fororal, parenteral or topical administration.
 2. A method for protectingcells in a subject from oxidative damage which entails administering tothe subject an effective amount of a compound which is(Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)aceticacid or a pharmaceutically acceptable salt thereof.
 3. A method ofpreventing or reducing the extent of tissue damage of an ischemic eventin a subject at elevated risk of an ischemic event, comprising: a.identifying a subject at elevated risk of an ischemic event; and b.administering a therapeutically effective amount of the compound(Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4, 5-trimethoxyb enzylidene)-1H-inden-3-yl)acetic acid, or a pharmaceutically acceptable saltor prodrug thereof to the subject, thereby preventing or reducing theextent of tissue damage of an ischemic event in a subject at elevatedrisk of an ischemic event.
 4. The method of claim 3, wherein the subjectpossesses one or more risk factors for an ischemic event selected fromthe group consisting of high blood pressure, heart disease, highcholesterol levels, sleep apnea, previous occurrence of stroke, smoking,excessive alcohol consumption and excessive weight.
 5. The method ofclaim 3, wherein the ischemic event is an ischemic event of a tissueselected from the group consisting of the heart, brain, kidneys andliver.
 6. The method of claim 3, further comprising administering to thesubject a therapeutically effective amount of a medicament selected fromthe group consisting of aspirin, a blood pressure medication, a type IIdiabetes medication and an mTOR inhibitor, optionally wherein the typeII diabetes medication is metformin, optionally wherein(Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)aceticacid, or a pharmaceutically acceptable salt or prodrug thereof and themedicament selected from the group consisting of aspirin, a bloodpressure medication, a type II diabetes medication and an mTOR inhibitorare co-administered to the subject.
 7. A method of treating a subjectwith retinitis pigmentosa, comprising administering to said subject atherapeutically effective amount of the compound (Z)-2-(5,6-dimethoxy-2-methyl-1-(3, 4,5-trimethoxybenzylidene)-1H-inden-3-yl)acetic acid, or apharmaceutically acceptable salt or prodrug thereof.
 8. A method ofinhibiting production of ROS in a cell, in vivo or in vitro, comprisingcontacting the cell with an effective amount of the compound(Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)aceticacid, or a pharmaceutically acceptable salt thereof.
 9. Amend asfollows: A method of protecting a normal cell against oxidative damage,in vivo or in vitro, comprising contacting the cell with an effectiveamount of the compound(Z)-2-(5,6-dimethoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)aceticacid, or a pharmaceutically acceptable salt thereof.
 10. Thepharmaceutical composition of claim 1, wherein The composition is in theform of a tablet, powder, granule, capsule, liquid, gel, syrup, slurry,solution or suspension.
 11. The pharmaceutical composition of claim 1,which is in the form of a solid.
 12. The pharmaceutical composition ofclaim 11, wherein the carrier comprises lactose, mannitol, corn starchor potato starch.
 13. Amend as follows: The pharmaceutical compositionof claim 11, wherein the solid composition comprises at least one of abinder, a disintegrant, and a lubricant.
 14. Amend as follows: Thepharmaceutical composition of claim 13, wherein the solid compositionfurther comprises at least one of a diluent, a buffering agent, amoistening agent, a preservative, and a flavoring agent.
 15. Thepharmaceutical composition of claim 1, which is in the form of an eyedrop or eye ointment.
 16. The pharmaceutical composition of claim 15,further comprising at least one of a buffering agent, an isotonizingagent, an antiseptic, a pH adjustor, a thickener, a chelating agent anda solubilizing agent.
 17. The pharmaceutical composition of claim 15,further comprising purified lanolin, white petrolatum, macrogol,plastibase or liquid paraffin.
 18. The pharmaceutical composition ofclaim 1, which is in the form of a liquid or semi-liquid.
 19. Thepharmaceutical composition of claim 18, which is in the form of aliniment, lotion, cream, ointment, paste, or drop.
 20. Thepharmaceutical composition of claim 19, which is in the form of an eyelotion.
 21. The pharmaceutical composition of claim 20, furthercomprising a sterile aqueous solution.
 22. The pharmaceuticalcomposition of claim 19, which is in the form of a lotion or linimentand further comprises at least one of an agent to hasten drying and tocool the skin, and a moisturizer.
 23. The pharmaceutical composition ofclaim 1, which is in the form of a semi-solid.
 24. The pharmaceuticalcomposition of claim 23, further comprising at least one of ahydrocarbon, a mucilage, an oil of natural origin, wool fat, a fattyacid, a surface active agent, and a suspending agent.
 25. Thepharmaceutical composition of claim 1, which is in a form suitable forparenteral administration.
 26. The pharmaceutical composition of claim25, wherein the carrier is aqueous.
 27. The pharmaceutical compositionof claim 26, wherein the carrier is a physiologically compatible buffer.28. The pharmaceutical composition of claim 25, which is in the form ofa suspension and the carrier comprises a lipophilic vehicle.